TW572772B - Compositions for use in hydrating an electrophoresis support to improve zone electrophoresis - Google Patents

Description

572772 V. Description of the invention (1) Zonal electrophoresis technology on agarose gel on the background of the present invention can separate protein components contained in samples, especially in biological samples, such as plasma, blood, Urine, cerebrospinal fluid, etc. In a gel-like medium containing a buffer solution, proteins deposited on the surface of the gel are ionized and move at different rates depending on the respective charges under the effect of the electric field. The thinness of the band obtained after electrophoresis, the component corresponding to the sample separated and the analytical capability of the step, are mainly determined by the thinness of the sample loading. Different types of sample applicators (ap p 1 i c a t 〇 r) can be used to improve the thinness of the load. This applicator is commonly referred to as a sample comb, and can take many different forms, as explained by the following device: "Groove" combs, where the comb teeth are grooved, and their edges are parallel to the plane of application, The device performs capillary action on a biological sample. The comb is then added to the gel under the effect of its weight. The comb teeth are then in contact with the surface of the glue, and the sample is transferred from the tooth comb to the glue. The comb teeth can be made of metal or plastic. The π-strip π comb has the same principle as the grooved comb, but its comb teeth include two parallel strips placed at the ends of a plane perpendicular to the applicator plane. The sample can be capillarized into the space defined by the two zones, and loaded on the glue in the same way, such as using a grooved comb. As before, the comb can be made of metal or plastic. The comb teeth of the film comb are composed of a microporous film. This comb is described in European patent EP-A-0 4 9 3 9 9 6 and US patent US-A-5 4 6 4 5 1 5 and US-A-5 4 0 5 5 16. The comb teeth are impregnated with the sample until the microporosity is thin. 572772 5. Invention description (2) The membrane is saturated. However, the surface of the glue contacts the spread of the glue and the different coatings are applied. When conducting electric (immunofix, usually, the dilution is measured from the second to 120 seconds as described above, the compression is reduced. This compression is reduced. Consider its recompression on the surface of the rubber surface to expose it during pressing while it is moving. The teeth of the comb, which are less stained or used by the present invention after being coated by the present inventor, are in the same manner as in the above two cases, and in this case, the sample is loaded with a protein from the microporous membrane. Applicator type When thin packs can be produced with more or less efficiency, the sample is usually diluted, especially for immunoimmobilization (ati ο η). The most commonly used dilutions are from 1/3 to 1/10. The solution is physiological water to keep the protein in the solution. Ground, within an average time of 30 seconds to 120 seconds, preferably 60, depending on the dilution of the applicator and the sample, after using the applicator to apply to the glue, the comb teeth contact the glue The position of is marked by zoom-in, which does not disappear during the movement. The amount of marking at the dyeing step that causes a lack of dyeing or the intensity of dyeing as described above (from a few grams to tens of grams, depending on the specific embodiment), this coating compresses the glue where the comb teeth contact the glue, Cause deformation in the form of shrink marks. If left unattended, this distortion persists and becomes visible after dyeing the gel, exposing the electrophoretic appearance. This deformation creates a zone that is unstained, which can be quite detrimental to the interpretation of the electropherogram. SUMMARY OF THE INVENTION The object of the present invention is to provide a device for excluding this loading mark regardless of the type of applicator. Loading markers-specific features generated on electrophoretic carriers have been observed

) 72772 V. Description of the invention Do not continue the sample loading area of the electrophoresis station on the gel, especially the package, and infiltrate; #! This office requires the body bands of the sample, heart: I-electrophoresis time of Suda electrophoresis Within the period of time, the rehydration period is' dilution from the inventor's diluent and shrinkage results in the first time when the compound is shifted to the compound labeled by the inventor's sample. Therefore, the composition of the sample of the present invention is negative, can be excluded or at least neutralized, so that it does not affect the interpretation of the subsequent result 'is applied in the sample applicator and the compressed band hydrates the electrophoretic carrier. This hydration must reach the zone of the electrophoretic carrier under conditions that allow the component-and the protein contained in the sample to be tested-for electrophoretic movement. In terms of being permeated onto the electrophoretic carrier and moving under an applied electric field, the electrophoretic carrier compressed by the applicator used to load the sample is rehydrated quickly and efficiently. For a certain period of time, the hydration must correspond to the time between 10% and 20% / Λ pair / the time when the voltage is applied to the electrophoretic carrier). "U Γ'water carrier plus voltage to time. Advantageously, do two or seven A plus 10/0 electrophoresis time plus voltage to time. τ It has been confirmed that some compounds have fish attached to them, so they are loaded on the electrophoretic carrier :, is it? :: 3 Imported sample The clear marks left are gone. This f depresses or at least weakens the electrophoretic carrier; 1: The entire movement t of the swimming movement continues to exist. And 5 already, and this private note is inflated at a position where these compounds have been observed. This causes pressure loss to provide a composition for electrophoresis on a carrier. Add β S 3 complex to the electrophoretic carrier with off-surface charge, its%, when the zone is loaded

Page 6 572772 V. Description of the invention (4) Hydration of the loading zone of the sample to be separated when the compression mark is caused by loading the sample. This compression is caused by the pressure of the applicator on the carrier. The presence of negative surface charges on the electrophoretic carrier is a non-zero electroosmotic (or electroosmotic flow) message. The compression of the electrophoretic carrier caused by loading the sample with the applicator can be visually observed and corresponds to the compression mark on the carrier. For example: When the pressure of the applicator is greater than 0.1 g / mm², this mark described as a V shape is usually less for an electrophoretic carrier composed of 0.8% agaric gum. At a depth of 100 microns and a width of about 1 mm between the two tops of V. Depending on the circumstances, the ionic compound used to prepare the composition of the present invention can be moved or or maintained almost immobile during electrophoretic movement. The lack of movement in the electric field is due to the nature of the compound, its concentration in the composition, or a combination of these factors. For a given electrophoretic carrier, when the electro-osmotic index (-rm index, relative movement index) is known, the concentration of ionic compounds present in the composition is determined as a function of the nature of these compounds, Especially its ability to move on the electrophoretic carrier after applying an electric field. This concentration must be selected so that the sample loading zone is hydrated on the electrophoretic carrier within the first time of the electrophoretic movement. As mentioned above, the desired hydration or rehydration effect of the electrophoretic carrier depends on the nature of the ionic compound used, and can also be affected by the composition of the electrophoretic carrier, especially its electro-osmotic index.

Page 7 572772 V. Description of the invention (5) In this respect, it is enough to add enough osmotic flow of 0. 07 or larger. Used in the present invention. It is selected from the group of the present invention that is not beneficial to the present invention during these electrophoresis (zwitterioni can be generated from two of the amino acids of the present invention as a real acid. In the formula of the present invention NH "R -C00H satisfies the above-mentioned combination with other compounds or non-ionic. The electrophoretic carrier is suitable for preparing when the electrophoretic carrier is agaric gel, in order to be able to retain water during the movement, especially on the gel compression belt, such as the use of non-zero gel For example, the ionic compound corresponding to the relative flow index -rm is dissolved to form the group defined above for a group of concentrations below its solubility limit. Detailed description of the preferred embodiment The first preferred embodiment In embodiments, the composition contains those ionic compounds that hardly move in an electric field. The compounds may rehydrate the compressed electrophoretic carrier tape, but move together with the components of the sample. The ground supply contains one or more selected from Zwitterionic c) compound composition. Examples of zwitterionic compounds cited in the content are: Zwitterionic compounds or zwitterionic buffers. For example, The composition of the invention can be an amine of the formula N Η 2-R _ C 0 0 Η where R comprises an uncharged polar branch. In another preferred embodiment, the amino acid is an amino acid selected from the group consisting of Wherein R contains a basic branched chain. One or other amino acids can be used alone or optionally mixed and used as a mixture. The compound can be an ion. When the carrier can be subjected to electro-osmosis, it can Examples of reusable amino acids in the composition of the present invention are: amines

Page 8 572772 V. Description of the invention (6) Acetic acid (The preferred concentration is compatible with its solubility in water at 25 ° C; in this respect, the concentration range that can be used is 1 mole to 3.33 Molar concentration) and phenylalanine at a concentration of 0.28 Molar. Other examples are selected from the group consisting of L-lysine at a superior concentration of 1 mole or more, preferably L-arginine at a concentration ranging from 0.45 mole to 0.86 mole and A preferred concentration is the amino acid of L-histidine at a concentration of 0.27 moles. Examples of zwitterionic buffers that can be cited for use in the present invention at concentrations greater than 0.3 Molar and within their solubility limits are the following biological buffers or their salts: TRICINE (N-ginseng (hydroxymethyl) Group) methyl-glycine); Hepis (1 ^? £ 8) to-(2-hydroxyethyl) piperazine-"-(2-ethanesulfonic acid); Pipes (PIPES) ( Piperazine-N-N'-bis (2-ethanesulfonic acid); Mopas (MOPS) (3- (N-morpholinyl) propanesulfonic acid); Tabos (TAPS) (N-gins (hydroxyl (Methyl) methyl-3 -aminopropanesulfonic acid); AMPSO (AMPSO) (3- ((1 ,: 1 -Diamido-2-hydroxyethyl) amino) -2-hydroxypropanesulfonic acid Acid); TES (T- (Hydroxy) fluorenyl-2-aminoethanesulfonic acid); BES (B, N-N-bis (2-hydroxyethyl) -2- Aminoethanesulfonic acid); BICINE (5N, N-bis (2-hydroxyethyl) glycine); CHES (CHEM) (2- (N-cyclohexylamino) ethanesulfonic acid) Acid); DIPSO (3- (N, N_bis (2-hydroxyethyl) amino-2-hydroxy-propanedioic acid); EPPS (N- (2- Hydroxyethyl) piperazine-N '-(3-propanesulfonic acid) Ma Pusuo (MOPSO) (3- (Ν- morpholino) propanesulfonic acid);

Page 9 572772 V. Description of the invention (7) Papso (POPSO) (piperazine-N, N'-bis (2'-hydroxypropanesulfonic acid); Mans (MES) (2-morpholinylethane In addition, the inventors have observed that the ideal rehydration effect of the electrophoretic carrier from the first time of sample movement can be obtained by ionic compounds that undergo electrophoretic movement, the limitation is that these compounds are present at a concentration sufficient to In addition to moving, the electrophoretic carrier is hydrated in the sample loading area. Such compounds are, for example, using high concentrations of salts, and in particular using routine procedures to determine the concentration and calculate the anion mobility. The lower the anion mobility The lower the effective concentration of salt. By way of example, these salts may be selected from NaCl, KC1 or NaHC03 at a concentration of 1 mole or higher, or borate at a concentration of 0.15 mole or higher, or 0.5 mole Ear or higher phosphate or acetate. The present invention therefore relates to the use of a composition that meets one or more of these definitions, for hydrating an electrophoretic carrier in a zone, wherein The sample is loaded. This application can use the composition of the present invention to dilute the sample to be separated by electrophoresis. The present invention also provides a kit for performing a method for separating sample components by electrophoresis, which is characterized by It consists of: • a composition as defined above; • an electrophoretic carrier composed of a gel with a non-zero electroosmotic flow. This kit may also contain any other reagents or devices, which are generally found in electrophoretic kits , Such as moving buffers, and indicators used to identify components separated from a sample by electrophoresis.

Page 10 572772 V. Description of the invention (8) The electrophoretic carrier that can be used in the present invention is generally a gel, and especially a gel having a non-zero electroosmotic flow. Examples that can be cited are agar gum, in particular HGT® or HEEO® agar gum from FMC-BIOWHITTAKER, or LE® or SHE® from HISTANAGAR, or Its mixture. These agar gums have non-zero electroosmosis. The agar gum used to prepare the electrophoretic carrier of the present invention has a concentration of 0.8% to 1% of agar, and may include a mixture of agar. The present invention also relates to a method for separating components of a sample by electrophoresis, including: • loading a sample using an applicator, and loading the sample at the same time as described above, and applying an electric field to move and display sample components The components are separated by electrophoresis. The display can be performed using staining and staining using antiviral serum. The methods defined above may be used and may specifically include immunoimmobilization steps. In particular, the method of the present invention is effectively used to detect the presence of a para-protein in a biological sample. This method can be advantageous in the zone when performing the method of the present invention, 'the first part that can be moved by electrophoresis can be separated on an electrophoretic carrier; the composition can be carried on the electrophoretic carrier; and in the case of immunoimmobilization, it can be routine The electrophoresis method is performed, and the components separated by electrophoresis are displayed. Out of the composition defined above, the pair in the right. The protein is sometimes reprinted :: Invention of

572772 V. Description of the invention (9) For simultaneous loading, the sample can be combined with the composition of the present invention before loading on the electrophoretic carrier, especially the sample is diluted in the composition. Additional features and advantages of the present invention will become clearer from the following examples: · Analysis of serum dual proteins diluted in a quenching solution (0 · 4 5 Molar concentration) on an immunofixation gel · (para ~ protein $ 〇 \ Va ^ ^ > 0 〇 · 3 2 grams of quenched solution was dissolved in 4 ml of water in a test tube. Therefore, the resulting dilution was ready to be used. The typed serum sample was diluted in the dilution solution. Dilute to one third for the lane corresponding to the electrophoretic pattern, such as IgA, IgM, IgK, and Ig λ lanes (for example, 30 micrograms of plasma and 60 microliters of dilution Solution), and the igG showed a one-sixth dilution in the narrow channel (for example: 20 microliters of plasma and 1000 microliters of dilution). ", 4 liters of physiological water was introduced into another test tube The same sample was diluted in the same way as above. 10 microliters of the diluted sample was fed into the European patent-A-0 4 9 3 9 9 6, the national patent-5 464 515 and the US patent-A- As described in 5 405 516: ϊ The individual wells of the applicator. 'Then this charged applicator is given 1 cent Apply to the surface of IF gel. 20 ί = The sample of the gel is used at 20 ° C. The instrument is adjusted at a temperature of about 10 minutes by electrophoresis using the self-power of a tile. The antiviral serum (anti-1gG, anti-1, anti-1 §M (several tens of cultures of each moving lane and the dual protein tyPed, and corresponding to the electrophoretic type of the lane has Wu fixed solution. Excess of Reagents are removed and the gel is dried

572772 V. Description of the invention α〇) ~ _, and stained with acid blue violet (acid vioiet). The samples diluted in quenching were observed on gels. Gels diluted with physiological water showed strong inscriptions. . ~ 'One one one. One-~ _ one one, ... "One .. one' ,,,,, and applicator print example No. 2: Analysis on immunofixation gel (Typ acid solution (0.86 Mo) Ear concentration) of serum dual protein) diluted in spermine (para-proteiη), 0.6 g of L-spermine acid was dissolved in a test tube and a 4 ml dilution was prepared for use. The typed serum was sampled in Because the solution is diluted to one-third, it is used to correspond to the electrophoretic type of “fresh”, such as IgA, IgM, IgK, and Ig λ slots (for example: 30 micron; 60 microliters of dilution ), And [gG] shows that the dilution in the narrow channel becomes six points (for example: 20 microliters of plasma and 100 microliters of dilution). 4 ml of physiological water is introduced into another test tube to Dilute the same sample in the same way as above. 10 microliters of the diluted sample is fed into European Patent-A-〇 4 9 3 9 9 6, US Patent-A-5 464 515 and US Patent-A-5 405 Into individual wells of the thin film applicator described in 516. The charged applicator is then applied to the surface of the IF glue in 丨 minutes. Apply to the IF glue These samples were separated by electrophoresis with 20 watts of electricity for about 10 minutes using an instrument adjusted at a temperature of 20 ° C. After movement, specific antiviral serum (anti-I g G, anti-I ga, anti- I g μ, anti-IgK, anti-Ig) to culture each moving lane and type the para-protein, and the lane corresponding to the electrophoretic type has

Page 13 572772 V. Description of the invention (11) '^-Protein fixation solution. Excess reagent was removed and stained with acid blue-violet. And ^ the knees were dry and dry. Samples diluted in arginine were observed on the gel, and those gels diluted in physiological water showed strong applicators.

Remember. At the time, I Na 2 Cao Er was analyzed on the Λ epidemic = fixed gel (typing) the serum dual-protein (para-prote in) diluted in sodium chloride cardiac fluid (1.2 Moore) °, 〇 · 29 grams of NaCl was dissolved in a test tube of 4 buckets of silk® into the supply & TJ zero liter of water. The resulting dilution is then ready for use. To be typed blood φ junior menstrual, -8-m month sample is diluted to one of two knifes in the dilution solution, which is used to correspond to the electrophoresis such as IgA, IgM, IgK and IgA slot (for example: 3 〇t: m = Rn as a liter of diluent) 'Sub- and narrowly dilute IgG for IgG (for example: 20 microliters of plasma and 丨 00 microliters of dilution) ,,,,,,,,,,,,,,,,,,,, 4 ml The physiological water was introduced into another test tube, and the same sample was diluted in the same manner. The upper phase of the U-million, 10 microliters of the diluted sample was fed into the European patent-A-〇 4 9 3 9 6, ΪΞΪΓ_Α_ 5 4 6 4 5 1 5 and the US patent-A-5 4 0 5 5 1 V Ϊ Ϊ tV! In individual wells as described in 6. This charged applicator was then applied to the surface of the IF gel in 1 minute. $ To IF glue for these samples was used at 20. . An instrument adjusted at a temperature of 20 watts is separated by electrophoresis for about 0 minutes. f. When moving, the specific antiviral serum (anti-IgG, anti-IgA, anti-IgM, Ig K, anti-Ig λ) is used to culture each moving lane to pair the proteins.

572772 5. Description of the invention (12) (para-protein) typed, and there is a protein fixation solution corresponding to the electrophoretic type in the narrow channel. The excess reagent was removed and the gel was dried and stained with acid blue violet. Samples diluted in concentrated NaCl solution were observed where they were loaded on the gel. No mark was shown, while those diluted with physiological water showed strong applicator marks.

Page 572772 Illustration

Claims (1)

572772, No. 901 (33911 Patent Application Amendment 1. A composition for a method for separating sample components by electrophoresis on an electrophoretic carrier, comprising an ionic compound that applies an electric field to an electrophoretic carrier having a negative surface charge. The resulting compression marks cause hydration in the sample zone to be separated. 2. A composition according to item 1 of the scope of patent application, wherein the ionic compound is not substantially moved in an electric field. 3. The composition according to item 1 or 2 of the patent application scope, wherein the concentration of the ionic compound is hydrated by the loading zone, so that its presence in the sample loading zone excludes the compression mark caused by loading the sample. 4. The composition according to item 1 or 2 of the scope of patent application, wherein the ionic compound is selected from zwitterionic compounds. 5. A composition according to item 4 of the patent application, wherein the zwitterionic compound is an amino acid. 6. A composition according to item 4 of the scope of patent application, wherein the zwitterionic compound is a zwitterionic buffer. 7. The composition according to item 5 of the patent application, wherein the amino acid formula is NH2-R_C00H, and R comprises an uncharged polar branch chain. 8. The composition according to item 5 of the patent application, wherein the amino acid formula is NH2-R-C00H, and R comprises a basic branched chain. 9. A composition according to item 7 of the scope of patent application, wherein the amino acid is aminoacetic acid and is present in a concentration compatible with its solubility in water at 25 ° C; or phenylpropylamine in a concentration of 0.28 mole acid. 10. The composition according to item 8 of the scope of the patent application, wherein the amino acid is selected from the group consisting of L-lysine at a concentration of 1 mole or higher, with a concentration range of 0.45. O: \ 69 \ 69338-920325.ptc Page 17 572772 _ Case No. 90103911 ^ Amended on the month of January_ Sixth, the scope of application for patents Molar concentration to 0.86 Molar concentration of L-arginine, and the concentration is 0.27 Molar concentration of L-histidine. 1 1. The composition according to item 4 of the scope of patent application, wherein the zwitterionic buffer is a biological buffer selected from the group consisting of: Trinicine (N-gins (hydroxymethyl) methyl-glycine) ; HEPES (N- (2-hydroxyethyl) piperazine-N '-(2-ethanesulfonic acid); PIPES (piperazine-N-N'-bis (2 -Ethane sulfonic acid); MOPS (3- (N-morpholinyl) propanesulfonic acid); TAPS (TAPS) Burnt lutein); AMPSO (3-((1, 1-dimethyl-2 -hydroxyethyl) amino) -2-hydroxypropanesulfonic acid); TES (N) -Ginseng (hydroxymethyl) methyl-2 -aminoethanesulfonic acid); BES (B, N (N, N -bis (2-hydroxyethyl) -2-aminoethaneethanesulfonic acid); Bayesin (BICINE) (5N, N-bis (2-hydroxyethyl) glycine); CHRIS (CHES) (2- (N-cyclohexylamino) ethanesulfonic acid); Dipso (DIPSO) ( 3- (N, N -bis (2-hydroxyethyl) amino-2 -hydroxy-propionate); Ips (£ 3) (1 (2-hydroxyethyl) piperazine-" -(3-propanesulfonic acid); Mapuxo (MOPSO) (3- (N- (Phenyl) propanesulfonic acid); Papso (POPSO) (piperazine-N, N'-bis (2'-hydroxypropanesulfonic acid); Mans (M ES) (2-morpholinylethanesulfonic acid) ); O: \ 69 \ 69338-920325.ptc Page 18 572772 _Case No. 90103911 _____ Sixth, the scope of patent application or the salts of these buffers. 1 2. The composition according to item 1 of the scope of patent application, further comprising a salt at a concentration sufficient to hydrate the compression zone of the electrophoretic carrier under the effect of an electric field before its removal. 13. The composition according to item 11 of the scope of patent application, wherein the concentration of the amphoteric buffer is greater than 0.3 Molar. 14. The composition according to item 12 of the scope of the patent application, wherein the salt is selected from the group consisting of NaCl, KC1 or NaHC03 with a concentration of 1 mole or higher, or a borate with a concentration of 0.15 mole or higher, Or a phosphate or acetate at a concentration of 0.5 mole or higher. 1 5. A method of hydrating an electrophoretic carrier in a loading zone of a sample to be separated by electrophoresis, the method comprising the step of using a composition according to item 1 of the scope of the patent application during electrophoresis. 16 — A method of diluting a sample separated by electrophoresis, the method comprising the step of adding the composition in the scope of patent application No. 1 to the sample to be analyzed. 17. A kit for performing a method for separating sample components by electrophoresis, comprising: • a composition according to item 1 of the scope of patent application; • a gel composed of a gel having a non-zero electroosmotic flow Electrophoresis vector. 1 8. The kit according to item 17 of the scope of patent application, wherein the electrophoretic carrier is agarose gel. 1 9. A method for separating components of a sample by electrophoresis, comprising: • loading a sample using an applicator, the components of the sample O: \ 69 \ 69338-920325.ptc Page 19 572772 _Case No. 90103911 Amendment_ VI. The scope of the patent application can be separated on the electrophoretic carrier; • At the same time, load the sample and load 1 item according to the scope of the patent application The composition is applied to the electrophoretic carrier; • An electric field is applied to move the sample components; 'show the components separated by electrophoresis. 20. The method according to item 19 of the scope of patent application, wherein the sample and the composition are combined before loading on an electrophoretic carrier. 2 1. The method according to item 19 or 20 of the scope of the patent application, wherein the electrophoretic carrier is an agar gelatin containing a negative surface charge. O: \ 69 \ 69338-920325.ptc Page 20